Model Logic

Protease inducible secretion

Retention of proteins in ER lumen was demonstrated by confocal microscopy and detection of the protein in the cell medium.
A variant of TEVp active in the ER lumen was implemented to control protein secretion from the ER lumen.
Retention of proteins on the ER membrane was also demonstrated by confocal microscopy and detection of the protein in the cell medium.
Rapamycin induced cleavage was used for controlled and inducible secretion of proteins from the ER membrane.

To achieve a fast regulated cellular response resulting in the release of a protein, we decided to mimic the release of insulin from beta cells where the protein of interest is pre-formed and present in the cell in secretory granules. In contrast to the specialized storage and release mechanism of insulin from beta cells we wanted to develop a more general and modular solution by making use of components already existing in different types of cells. Additionally, there should be minimal leakage from the protein depot in the uninduced state and after induction secretion from the cell should be fast.

Further explanation ...

Not many systems for the inducible release of proteins have been engineered to date. In one of the few examples Rivera et al. developed a system where the protein of interest was fused to a conditional aggregation domain (CAD). Rivera2000 . These domains form aggregates in the endoplasmic reticulum (ER) that are too large to exit the ER. After the addition of a small synthetic molecule, the CADs start to disaggregate and the protein of interest can be secreted. In the second example Chen et al. introduced a light-triggered secretion system. They also based their system on conditional aggregation; however they used the plant photoreceptor UVR8 which forms photolabile homodimers to make aggregates on the ER membrane. Upon light excitation the aggregates made by UVR8 started to disaggregate and were transported from the ER to the plasma membrane, but have not been observed in the cell supernatant. Chen2013 The weakness of the two described systems is that they both rely on the exogenous chemical or physical signals instead of using a biochemical signal to induce the secretion, which means that they can’t be integrated into the signaling system that’s senses the cellular state. In order to better respond to the state of the cell or a logic circuit inside a cell we decided to develop an inducible secretion system based on the biochemical signal.

Many proteins that reside on the membrane or in the lumen of the ER contain short peptide signals. Proteins present in the lumen of the ER contain a KDEL C-terminal sequence (Lys-Asp-Glu-Leu) while type I transmembrane (TM) proteins contain a dilysine (KKXX) motif on their C-terminus (cytosolic side). Munro1987, Jackson1990, Stornaiuolo2003 . The mechanism that allows these proteins to stay in the ER is more retrieval than retention. However we decided to use the term retention for description of this process. ER luminal proteins interact with the KDEL receptor, a transmembrane ER resident protein. The cytosolic part of the KDEL receptor interacts with coat proteins I (COP I) which coat vesicles and are responsible for transporting proteins from the cis end of the Golgi apparatus (cis-GA) back to the ER. The KKXX motif present on type I TM proteins can directly interact with the COP I for retrieval. Stornaiuolo2003, Letourneur1994 .

Our idea was that if we proteolytically remove the retention signal, the protein of interest would no longer be retrieved back to the ER and could be secreted from the cell. To achieve this we designed two types of secretory reporters, one type based on the luminal retention using KDEL sequence and the other based on the transmembrane retention with a KKMP sequence. In each case, the retention sequence was preceded by a TEVp cleavage site to allow for inducible secretion, which could be replaced by any other peptide target of our orthogonal protease set.

Results

Secretion from the ER lumen

To achieve and detect the inducible secretion from the ER lumen, we created two reporter constructs with a cleavable KDEL sequence targeted to the ER lumen: SEAP^KDEL and TagRFP^KDEL. Those proteins contained a protease target motif between the reporter domain and the KDEL domain, aimed to enable protein secretion after the proteolytic cleavage. We used a TEVp variant (erTEVp) for all of our experiments with luminal retention.

Further explanation ...

In order to rely on TEVp cleavage in the ER lumen, we had to take some additional considerations into account. Cesaratto et al. Cesaratto2015 reported that the wild type TEV protease is not active in the lumen of ER. They designed a TEV protease variant active in the endoplasmic reticulum by preventing two major types of post-translational modifications: N-glycosylation and cysteine oxidation. To avoid these inhibiting modifications, mutations N23Q, C130S and N171T were made. To ensure correct localization and accumulation of this TEVp variant inside the endoplasmic reticulum, we also attached a signal sequence at the N-terminus and KDEL at the C-terminus of the protein.

**Cleavage with ER-residing protease (erTEV) facilitates secretion of reporter from cells.**

(A) Scheme of the reporter with cleavable KDEL retention signal and protease target motif. (B) The reporter with the KDEL retention signal was localized in the ER. HEK293T cells were transfected with the indicated reporters and in (C) also with erTEVp. Localization was detected with confocal microscopy. (C)The reporter was detected in the medium of cells only when cotransfected with erTEVp. HEK293T cells were transfected with the indicated constructs. Reporters were detected with WB in the concentrated medium.

When the TagRFP^KDEL reporter ( 1 A) was expressed in the ER without an active erTEVp we confirmed its localization in the ER with confocal microscopy ( 1 B). Additionally, we could not detect any TagRFP in the cell medium with Western blotting. When erTEVp was present and active in the ER, the KDEL sequence was removed from the reporter and the protein was secreted from the cell, which we detected with Western blot ( 1 C), demonstrating that proteolytic activity in the ER can regulate protein secretion.

Using SEAP^KDEL we were able to confirm that the reporter is not present in the cell medium without coexpression of erTEVp. When erTEVp was cotransfected with the reporter, we detected a large increase in enzymatic activity in the medium ( 2 ).

**Secretion of the SEAP reporter from ER lumen by cleavage with ER-resident protease.**

HEK293T cells were transfected with indicated reporter and erTEVp. Increased SEAP activity was detected in the medium of cells expressing both reporter and erTEVp protease.

Secretion from the ER membrane

The second approach to regulate protein secretion from the ER by protease was to used KKMP ER retention peptide linked to the transmembrane protein with a protease target motif on the cytoplasmic side, N-terminal to the KKMP peptide. A transmembrane (TM) domain from the B-cell receptor (Bba_K157010) was used for the integration of target proteins in the ER membrane. Similar as described above, two reporter constructs with SEAP and TagRFP (SEAP:TM^KKMP and TagRFP:TM^KKMP) were designed and the constructs also contained a signal sequence at their N-terminus and a proteolytically cleavable ER retention signal at their C-terminus. In case of the transmembrane targeted reporters we used the KKMP retention signal preceded by 3 copies of the TEVp cleavage site on the cytosolic side of the membrane.

Additionally, either one or four furin cleavage sites were inserted between the protein of interest on the luminal side of the ER, which enable cleavage of the reporter protein from the membrane, but this could occur only after the KKMP had been removed and the protein could enter the trans-GA. Furin is a native cellular endoprotease that is active only in the trans-GA.Henrich2003 This allowed us to design our constructs so that they are cleaved off of the membrane without any modified scar sequences attached to them.

**Localization of protease-responsive reporters on ER depending on the proteolysis.**

(A) The transmembrane reporter without the KKMP retention signal was localized both on the ER and plasma membrane. (B) The transmembrane reporter with the KKMP retention signal was localized exclusively on the ER membrane. (C) After cleavage of the KKMP retention signal, the transmembrane reporter translocated to the plasma membrane. HEK293T cells were transfected with the indicated reporters and in (C) also with TEVp. Localization was detected with confocal microscopy. Each image is accompanied with a scheme of the transfected construct. (D) Glycosylated reporter was detected in the medium of cells transfected with the transmembrane reporter without the KKMP retention signal. HEK293T cells were transfected with the indicated constructs. Reporters were detected with WB in the concentrated medium. In lane 2, sample was incubated with N-glycosidase F.

**Inducible secretion of reporter localized on ER membrane.**

SEAP activity was increased in the medium of cells induced with rapamycin. (B) Scheme of the transmembrane reporter with cleavable KKMP retention signal and inducible protease. HEK293T cells were transfected with the indicated reporter and rapamycin inducible split proteases. Uncleaved proteases were used as positive control.

Localization of the TagRFP:TM^KKMP reporter was confirmed by the confocal microscopy. We used a control reporter without the KKMP retention signal (TagRFP:TM) which we detected both on the ER and the plasma membrane ( 3 A). In case of the present KKMP retention signal, the reporter was detected only on the ER ( 3 B). When TagRFP:TM^KKMP was coexpressed with TEVp, localization of the reporter was similar to the localization of the positive control (TagRFP:TM) on the plasma membrane and the ER ( 3 C).

A band with a slightly larger apparent size than the expected size of TagRFP (28 kDa) was detected by western blotting in cells transfected with TagRFP:TM. We showed that the unexpected difference in size was due to glycosylation, as we detected the protein at the expected size after deglycosylation of the medium sample with N-glycosidase F. We were unable to detect a corresponding band in the medium of cells transfected with TagRFP:TM^KKMP in the absence of the protease.

Together, these results confirm that localization and secretion of the protein reporter with the transmembrane domain depends on the presence and proteolysis of the KKMP retention signal and that proteolysis can be used to induce secretion of already synthesized protein.

Finally, we cotransfected cells with SEAP:TM^KKMP and rapamycin-inducible split TEVp. We detected increased levels of the SEAP enzymatic activity in the medium of cells stimulated with rapamycin, which was dose dependent with respect to the amount of the transfected reporter-coding plasmid ( 4 ). These results confirm that secretion of a target protein can be made inducible by an externally supplied signal, processed through our split protease system.

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Model of a two state inducible system based on autoinhibitory coiled coil interactions was designed.
Range of $Kd_B$ and $Kd_b$ values resulting in optimal signal to noice ratio was determined.

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Logic operations in biological systems have been tested with several approaches <x-ref>Singh2014</x-ref> . Our project relies on the reconstitution of split protein promoted by coiled-coil (CC) dimerization. The interaction between CC peptides can be finely tuned <x-ref>Woolfson2005, Gradisar2011, Negron2014</x-ref> , thereby CCs offers a flexible and versatile platform in terms of designing logic operation in vivo. With the purpose of understanding the relation that underlies the interaction between coiled-coil peptides and therefore using them in logic gates, we designed the following model ( ^[1] ). Our system is based on constructs that have been characterized in mammalian cells in the context of <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Logic">logic function design</a>. Two orthogonal CC segment, A and b, fused together in on chain can bind each other and form a stable CC pair. This complex exists in combination with the peptide B, which can also bind the peptide A and has a different affinity from the peptide b. The linker that connects A and b can be cleaved by a generic protease (e.g. TEVp), this irreversible reaction shifts the equilibrium towards a state in which all of the three peptides are free in solution and therefore compete for binding. In our experiments, a similar system as the generic coils A and B was fused to the <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters#cle">split reporter firefly luciferase</a>.

                           <figure data-ref="5.4.1.">
                               <img
                                       src="">
                               <figcaption> Scheme representing the CC interaction model

The two state system is considered at inducible by activity of TEV protease and signal both before and after cleavage is represented as reconstitution on split firefly luciferase reporter.

                               </figcaption>
                           </figure>

The relationship between the signal before and after cleavage by proteases is represented by the difference [AB] - [AB-b]. In order to understand the optimal combination of dissociation constant required to obtain a good signal we solved two systems of equations set up considering the two state of the reaction scheme, Before cleavage and After cleavage, (1) and (6) respectively, as separate phases of the reaction and additionally, considering cleavage as an irreversible and complete reaction.

Given values for total concentrations and Kd (from 10^-9 to 10^-3 M) the equations, for the reaction constants (2), (3) and (7), (8) and and for mass conservation (4), (5) and (9), (10), (11) were solved for the species at equilibrium.

                       Before cleavage
                       \begin{equation}
                       \ce{Axb + B <=>[Kd_x] A-b + B <=>[Kd_B] AB-b}
                       \end{equation}
                       \begin{align}
                       Kd_x &= \frac{[A-b]}{[Axb]} \label{1.1-2}\\
                       Kd_B &= \frac{[A-b] * [B]}{[AB - b]} \\
                       c_B &= [B] + [AB-b]\\
                       c_A-b &= [A-b]+[Axb]+[AB-b] \label{2.1-2}
                       \end{align}
                       After cleavage
                       \begin{equation}
                       \ce{Ab + B <=>[Kd_b] A + b + B <=>[Kd_B] AB + b}
                       \end{equation}
                       \begin{align}
                       Kd_b &= \frac{[A] * [b]}{[Ab]} \label{1.3-4}\\
                       Kd_B &= \frac{[A] * [B]}{[AB]} \\
                       c_A &= [A]+[AB]+[Ab]\\
                       c_B &= [B] +[AB]\\
                       c_b &= [b] + [Ab] \label{2.3-5}
                       \end{align}

                       The two systems are connected by the relation between the dissociation constants $Kd_b$ and
                       $Kd_x$,
                       \begin{equation}
                       Kd_x = Kd_b * 4 * 10^{-3} M^{-1}
                       \end{equation}
                       This relation approximates the higher affinity between the coils A and b when they are
                       covalently
                       linked by a short peptide (as in the system “Before cleavage”)
                       <x-ref>Moran1999, Zhou2004</x-ref>
                       .

The results have been plotted varying the $K_d$ for the interaction of A with both B and b, against the difference [AB] - [AB-b], where [AB] is considered the signal after cleavage and [AB-b] the signal before cleavage (leakage). The system revealed that in order to obtain a high difference between signal and leakage a high affinity of the coil B for the coil A (low $Kd_B$) is required, while on the other hand an excessive destabilization of the autoinhibitory coil b (high $Kd_b$) would prevent the signal to be visible ( ^[2] ).

                           <figure data-ref="5.4.2.">
                               <img
                                       src="">
                               <figcaption> Difference between [AB] and [AB-b] depending on the ratio of Kd
                                   values.

The plots display the difference (M) between the signal before after and the proteolytic cleavage (left) and the concentration of the species responsible for leakage [AB-b] (right) in a range of different Kd values.

                               </figcaption>
                           </figure>

This relationship suggested to try using a different version of the coiled-coils available in the toolset already used by the <a href="https://2009.igem.org/Team:Slovenia">Slovenian iGEM 2009 team</a> <x-ref>Gradisar2011</x-ref>. In order to obtain a detectable signal for <a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Logic">logic operation in vivo </a> we decided to use an inhibitory coiled-coil, which would be displaced by the second coiled-coil with higher affinity, only once is cleaved off its partner ($ Kd_B \lt Kd_b $). In doing so we selected P3 as B and P3mS as b, these two coiled-coil peptides present only few substitutions and the higher solubility of P3mS (b), which presents Gln and Ser instead of Ala in b and c position of the heptads, would favour the dissociation. We also tried differently destabilized versions of P3 and it turned out that, as in the forehead described model, an excessive destabilization (obtained by substituting a and d positions with Ala) leads to a small difference of the signal before and after cleavage. Using a slightly destabilized coiled-coil (P3mS-2A), which presents only 2 alanines in the second heptad, the signal after cleavage reached its maximum of 16 folds (<a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Logic#autoinhibitory">Logic Figure 10</a>).

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↑ 5.4.1.
↑ 5.4.2.

[1] 5.4.1.

[2] 5.4.2.

[1]

[2]

Team:Slovenia/Demonstrate

Protease inducible secretion

Results

Secretion from the ER lumen

Secretion from the ER membrane

References

Coiled-coil interaction model

References